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Experiments were performed to define the mechanism of intestinal absorption of dodecyl sulfate (DS), an amphipathic organic anion whose chemical structure resembles that of dodecanoate, a C12 fatty acid anion. With jejunal segments perfused in single-pass fashion in the anesthetized rat, steady-state absorption of DS was concentration dependent, with the apparent permeability constant (P(app)) ranging from 4 to 22 x 10(-5) cm/s. When DS concentration was held constant and net water absorption was induced by decreasing perfusate osmolality, DS absorption increased in direct proportion to water absorption, suggesting absorption by solvent drag via the paracellular route. However, DS absorption continued even when water secretion was induced by a hypertonic perfusate. Consequently, for all experiments, DS absorption could be empirically described as the sum of two terms: 1) absorption in the absence of water absorption (P(app) = 5.6 x 10(-5) cm/s) and 2) absorption induced by water movement [(delta P(app)/delta water absorption) = 0.2 x 10(-5) cm x s(-1) x microl segment(-1) x min(-1)]. In a polarized epithelial monolayer of renal epithelial cells (Madin-Darby canine kidney cells), DS was absorbed predominantly by a paracellular pathway, as the absorption rate increased threefold when paracellular junction pore size was increased by the addition of cytochalasin D. The calculated apparent radius was 2.9 A, indicating that the cross section of the molecule, not its length, determined the rate of absorption. It is concluded that absorption of DS in the intact animal occurs slowly and mostly via the paracellular route, because the fixed negative charge on the molecule retards rapid passive entry into the enterocyte, as occurs with protonated fatty acids. That absorption of DS persisted despite net water secretion suggests a low level of transcellular absorption across the jejunal enterocyte also occurs.

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowly than remnants derived from triolein emulsions. The effect associated with a saturated acyl chain at the glycerol 2-position could be reproduced by incorporating 2-stearoylglycerol (MS) in a triolein emulsion. When MS solubilized with rat albumin or in plasma was injected before the injection of a triolein emulsion, clearance of the triolein emulsion was unchanged. The metabolic fate of MS, monitored with 14C-labelled MS, was similar whether incorporated in triacylglycerol emulsion or injected independently. More than 95% of MS had disappeared from the circulation by 5 min after the injection and the radioactivity was found in liver, spleen, muscle and adipose tissue. Some MS label appeared in plasma triacylglycerol. Remnants made in vitro by incubating triolein or OSO emulsions with post-heparin plasma showed no differences in their disappearance from plasma. With OSO emulsion, the in vitro remnants were found to contain more MS than remnants made in vivo in hepatectomized rats. Simultaneous injections of mixtures containing OSO and triolein emulsions, or triolein emulsions with and without MS, each labelled with either [3H]cholesteryl oleate or [14C]cholesteryl oleate showed consistently slower remnant removal and decreased liver uptake of the emulsions containing OSO or MS. Affinity columns and immunodiffusion all indicated that there was no difference in the amounts of apolipoprotein E associated with OSO or triolein particles. The protein spectra of in vivo remnants derived from OSO and triolein emulsion were also similar when examined by SDS-PAGE and isoelectric focusing gels. Our results show that the effects due to OSO or MS are mediated by the presence of MS in the emulsion particle surface, while indirect effects expressed in plasma or liver are excluded. The precise mechanism of the effect remains to be established, but it does not correlate with measurable changes in the spectra of apolipoproteins associated with the emulsion remnants.

1. Two aspects of cholesterol absorption; (a) the importance of solubilization and (b) the effects of different bile salts on the mucosal metabolism and lymphatic output of cholesterol, have been investigated using two different in vivo techniques. 2. Bile diverted lymph fistula rats were infused intraduodenally at a steady rate with a constant lipid mixture containing labelled cholesterol, labelled oleic acid and mono-olein. The lipids were completely solubilized in either bile salts or a non-toxic non-ionic detergent, Pluronic F68. Labelled fatty acid was efficiently absorbed from either micellar infusate but virtually no labelled cholesterol appeared in the lymph in the absence of bile salts. 3. Short-term perfusions of the intestine of anaesthetized rats with the same micellar perfusates as above showed approximately 20% of the labelled cholesterol in the mucosa after 30 min perfusion with the bile salt micellar solutions. When the non-ionic micelles were used virtually no isotopic cholesterol left the lumen. 4. Mucosal uptake of labelled cholesterol was linearly dependent on the concentration of solubilized cholesterol in the perfusate and was not dependent on the bile salt concentration. 5. After 30 min the total amount of perfused isotopic cholesterol was recovered from either the lumen or the mucosa, but some fatty acid appeared to have been transported away from the mucosa by this time. 6. The initial rate of mucosal uptake of labelled cholesterol was similar from micellar perfusates using either taurocholate, taurodeoxycholate or taurofusidate. In contrast, after 8 h of infusion, lymphatic output of labelled cholesterol was markedly greater with taurocholate. 7. The increased lymph output with taurocholate was associated with an increase in the esterified fraction of both labelled and unlabelled cholesterol. Fatty acid was absorbed and esterified equally from all three types of perfusate. 8. These results suggested that for the first step in cholesterol absorption, viz. uptake from the lumen, solubilization by a planar detergent was essential. After uptake, the more rapid transfer of cholesterol to lymph in the presence of trihydroxybile acids appeared to be related to a more efficient esterification of cholesterol, but not to a more efficient resynthesis of triglyceride, the other major component of lymph chylomicrons.

Lipid emulsion particles were prepared by sonicating four different lipid mixtures (triacylglycerol (TAG), 70%; phospholipid, 25%; cholesteryl oleate (CO), 3%; and free cholesterol, 2%), then purified by density gradient ultracentrifugation. For three test mixtures, the TAG contained 50, 75, or 100% 1,3-dioleyl-2-stearylglycerol (OSO) with the remainder being triolein (OOO); 100% triolein in the lipid mixture was used as the control. After intravenous injection of the lipid particles into unanesthetized rats, removal of radioactive TAG fatty acid and CO from plasma was measured for 30 min, then liver and spleen uptakes were measured. When emulsions contained 75% or 100% OSO as TAG, the plasma removal rates of CO were, respectively, 60% or 30% of the rate when the TAG was 100% triolein; smaller recoveries of CO were found in the liver. The clearances of TAG fatty acid did not differ significantly and the recoveries of TAG fatty acid in the organs were not affected by the type of emulsion injected. Remnant particles were derived from donor rats in which uptake was blocked by exclusion of liver and other viscera from the circulation before injection of 100% OOO and 100% OSO emulsions. When injected into recipient intact rats, the removal of remnants from plasma was slower for remnants derived 15 min after injection of 100% OSO emulsions than from 100% OOO emulsions, showing that the slower removal of emulsion CO was due to slower remnant uptake from the plasma with OSO emulsions.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was undertaken to assess the role of luminal lecithin in the lymphatic transportation of fat as chylomicrons. Two doses of fat, the low and high dose, were fed to two different groups of rats, control and bile fistula. At low dose, infusing at 35 μmoles of total fatty acid per hr of a mixture of oleic acid and monoolein, molar ratio 2:1, solubilized in 55 μmoles of sodium taurocholate, there was no difference in the lymphatic output of absorbed fat during steady state (7th and 8th hour) absorption. Infusing at a high dose, 173 μmoles of total fatty acid per hr of a mixture of oleic acid and monoolein, molar ratio 2:1, solubilized in 55 μmoles of sodium taurocholate, the bile fistula rats had lower triglyceride and phospholipid output, with a higher proportion of oleic acid in lymph lecithin than did control rats. These alterations in bile fistula rats returned to normal by adding 10 μmoles per hr of biliary lecithin to the infusate. We conclude that intraluminal biliary lecithin plays a significant role in the translocation of high doses of absorbed fat into lymph and in the amount and type of lecithin synthesized.

1. 1. The aim of the experiments was to test in vivo whether absorption of lipid varied with the concentration solubilized in the lumen. Such a dependence has been demonstrated for everted sacs in vitro. 2. 2. Two types of perfusates were prepared in which the total concentration of absorbable labelled lipid and of conjugated bile acid was the same. In one type, most of the lipid was in micellar solution; in the other type, the solubilized concentration was lowered by adding an unabsorbable oil, glyceryl triether, into which micellar lipid was partitioned. 3. 3. Absorption was tested in rats prepared in two ways: (A) unanaesthetized with common bile duct and thoracic lymph duct cannulated in which lipid was continuously perfused by duodenal cannula; (B) anaesthetized with the small intestine cannulated proximally below the entrance of the common bile duct and distally in the lower third of the ileum. In Preparation B, the whole perfusate was injected at zero time and an even exposure to the entire mucosa ensured by to and fro displacement. 4. 4. In Preparation A absorption of [ 14 C]oleic acid and glyceryl[ 3 H]monooleyl ether into the lymph was the same for both lipids and both perfusates. Lipid was almost completely absorbed before the perfusate reached the distal half of the small intestine. 5. 5. In Preparation B disappearance of lipid from the lumen and uptake by the mucosa was less for the perfusate in which the concentration solubilized was lower. Oleic acid was absorbed more rapidly than monoether, relative to the concentrations solubilized. These findings agreed with results for everted sacs in vitro. 6. 6. It is suggested that in Preparation A decreased absorption per unit area can be compensated by utilization of more of the total absorptive area. In Preparation B and in everted sacs, no such compensation is possible and variation in absorption rate with solubilized concentration can be readily demonstrated.

Lymph fistula rats with either biliary fistula or pancreaticobiliary fistula were used to measure coenzyme A-independent mucosal cholesterol esterifying activity in the presence and absence of pancreatic exocrine secretion. Efficiency of pancreatic diversion was verified to minimize contamination of mucosal homogenates with adherent luminal enzyme. The synthetic activity of cholesteryl ester hydrolase (E.C. 3.1.1.13) was measured directly in mucosal homogenates. Indirect evidence for mucosal esterifying activity was obtained from hourly cholesteryl ester output into lymph when other factors known to affect cholesterol absorption were controlled. Rats infused intraduodenally at a constant rate with different concentrations of bile salts, polar lipid, and [3H]cholesterol showed that the infused [3H]cholesterol was absorbed and esterified with equal efficiency in the presence and absence of pancreatic flow. Total lymph output of free and esterified endogenous cholesterol was slightly less efficient in the pancreaticobiliary fistula group (85% of bile fistula values) but percent esterification was the same for both groups. Infusion of lipid-free micellar bile salts separately from other bile components produced a highly significant increase in absorption and esterification of lymph cholesterol for both groups. No correlation was found between cholesterol esterifying activity in a) the lumen or b) the mucosa, and cholesteryl ester output into lymph. The present study suggests an alternative enzyme dependent directly or indirectly on the presence of micellar bile salts in the lumen to explain intestinal cholesterol esterifying activity during absorption.

A luminal supply of biliary phosphatidylcholine is important in the translocation of absorbed fat into lymph and in the amount and composition of phosphatidylcholine concurrently synthesized. This study was undertaken to determine whether the effect was due to absorbed lysophosphatidylcholine, to a specific (1-palmitoyl) biliary lysophosphatidylcholine or to extra choline supplied by lysophosphatidylcholine. Rats with bile fistulae and thoracic duct lymph fistulae were given test meals of oleic acid and monoolein (molar ratio 2:1) infused duodenally for 8 h. Addition of choline chloride to the test meal increased lymphatic output of triglyceride and phospholipid but not to values found previously in rats with supplements of bile phosphatidylcholine or with bile ducts intact. Addition of dioleoyl phosphatidylcholine increased triglyceride and phospholipid output to values found in rats with intact bile ducts. Since dioleoyl phosphatidylcholine was as efficient as biliary phosphatidylcholine it was concluded that a luminal supply of 1-palmitoyl lysophosphatidylcholine was not essential. It seemed likely from the smaller effect of supplemented choline and from the fatty acid composition of lymph phosphatidylcholine that the essential requirement was a supply of absorbed lysophosphatidylcholine for rapid reacylation to phosphatidylcholine.

The importance of the bile acid structure on mucosal uptake and lymphatic absorption of cholesterol was investigated using four different taurine-conjugated bile acids. Pure synthetic conjugates of a trihydroxy bile acid, taurocholate, and three dihydroxy bile acids, tauroursodeoxycholate, taurochenodeoxycholate, and taurodeoxycholate were used to completely solubilize [14C]cholesterol and polar lipids for steady rate intraduodenal infusion for 8 hr in bile fistula rats. Lymph output and esterification of [14C]cholesterol and endogenous cholesterol were measured in hourly samples. A second group of bile fistula rats was given the same bile acids as the first group but without added cholesterol or other lipid, i.e., fasting lymph fistula group. Mucosal uptake of [14C]cholesterol was studied using recovery of [14C]cholesterol from lumen and mucosa after 1-hr infusions in conscious bile fistula rats. Lymph output of [14C]cholesterol was promoted more rapidly with taurocholate than with the dihydroxy conjugates and [14C]cholesterol output differed for the three groups given dihydroxy bile acids. The mass of cholesterol in lymph, measured chemically, varied in parallel with [14C]cholesterol absorption. For fasting lymph, infusion of dihydroxy bile acids failed to produce a significant change in endogenous cholesterol output when compared with rats given saline only. Taurocholate infusion markedly increased endogenous cholesterol in lymph of fasted rats. Under all conditions where cholesterol output was stimulated, the increase could be accounted for mainly as esterified cholesterol. Mucosal uptake of [14C]cholesterol during 1-hr infusions in conscious bile fistula rats was slower with the dihydroxy bile acids than with taurocholate. The results indicate the marked effect of the number and configuration of the hydroxyl groups on the solubilizing bile acid for cholesterol absorption.

The Ca2+ and Mg2+ contents of strips of the circular smooth muscle from the stomach of the toad (Bufo marinus) were measured after varying lengths of exposure to NaCl-Ringer and K2SO4 depolarizing Ringer solutions in which the Ca2+ and Mg2+ concentration was varied. The Ca2+ content of the smooth muscle was correlated with the contractility of the muscle when the tissue was first depleted of Ca2+ to abolish responses and then controlled replacements made. In Ca2+-free depolarizing K2SO4-Ringer the Ca2+ content fell from 0.95 ± 0.02 (mean ± S.E. of mean) to 0.14 ± 0.01 μmole per g wet weight before contractility to acetylcholine was lost. When only 9% of the tissue Ca2+ was replaced by exposure to 0.08 mM Ca2+ in the Ringer solution, full contractility was restored, the muscle then containing 0.23 ± 0.01 μmole per g wet weight. However, contractions produced by alternating current applied in the longitudinal direction of the fibres were practically eliminated when the tissue still contained 0.31 ± 0.01 μmole of Ca2+ per g wet weight. 26% of the maximal contractility was restored when the tissue contained 0.43 ± 0.01 μmole per g wet weight and 42% response when the tissue contained 0.53 ± 0.02 μmole per g wet weight. When the tissue contained supranormal amounts of Ca2+, the contractile response to maximum electrical stimulus equalled that produced by a maximal dose of acetylcholine. The experiments suggest that acetylcholine response depends on a small fraction of Ca2+ located in the cortex of the cell while the effect of alternating current is to displace Ca2+ from myoplasmic sites.

1. 1. Uptake and efflux of labelled micellar cholesterol by everted sacs of rat jejunum was compared for two detergents, (a) pure sodium taurocholate-sodium taurodeoxycholate (4:1, on molar basis) and (b) a high-molecular-weight non ionic detergent, Pluronic F68. Oleic acid and monoolein were also incorporated in the micellar solutions. 2. 2. Uptake of cholesterol from bile salt micelles was 2–4 times faster than from non-ionic micelles, for incubation periods varying from 10 min to 2 h. 3. 3. Uptake from bile salt micelles increased with temperature (apparent activation energy about 6 kcal/mole cholesterol) but uptake from non ionic micelles did not. Metabolic inhibitors had little effect. 4. 4. When sacs were loaded with isotopic cholesterol from bile salt micellar media, subsequent efflux into buffer, or into unlabelled cholesterol in non-ionic micelles or in bile salt micelles was slower than was efflux into a given medium after loading with isotopic cholesterol from non-ionic micelles. On the other hand, efflux into bile salt micellar medium was faster for a given loading medium than efflux into non-ionic micelles. 5. 5. It is suggested that cholesterol uptake may involve two stages only one of which is temperature sensitive and is promoted by bile salts.

1. 1. Everted sacs of rat jejunum were used to measure the effect on uptake of labelled oleic acid, 1 mM, when an unabsorbable oil (glyceryl-trioleyl ether) was present. The incubation medium also contained monoolein 1 mM, and pure bile salts (sodium taurocholate-sodium taurodeoxycholate, 4:1 on a molar basis). 2. 2. Increasing concentrations of triether had no effect on the low uptake of oleic acid in the absence of a micellar phase, bile salts 1 mM, when virtually all the lipid was in emulsified particles. 3. 3. When most of the oleic acid (and monoolein) was present in a micellar phase, bile salts 4 mM or 5 mM, uptake was greatly accelerated. Addition of increasing amounts of triether now led to partition of an increasing proportion of oleic acid into the emulsified oil particles with a corresponding reduction in concentration of oleic acid in the micellar or isotropic phase. Uptake of oleic acid was reduced in linear proportion to reduction in micellar concentration. 4. 4. The regression of oleic acid uptake on isotropic aqueous concentration was the same in these experiments, in which bile salt concentration remained constant, as in previous experiments in which the isotropic concentration was varied by altering the bile salt concentration. 5. 5. These experiments provide further evidence that uptake in vitro depends on the concentration of fatty acid in the isotropic aqueous phase, and not on the concentration of bile salts per se .

Summary The effect of aminopterin on fat absorption into the lymph of unanesthetized rats has been studied at various times after the administration of the drug. A consistent effect on fat absorption has been shown, and this has been related to the age of the mucosal cells. After inhibition of mitosis with aminopterin, fat absorption remains normal for at least 32 hr, and then there follows a period of severe malabsorption which lasts for 2 days. This malabsorption has been shown to be mucosal in origin. Mitosis in the crypts recommences on the fourth day after aminopterin, but fat absorption does not begin to recover until about 24 hr later. The results indicate that a preliminary correlation of mucosal cell age to fat absorption is possible. Newly arisen cells appear to be unable to absorb fat until they are about 24 hr old, and then they retain this ability until they are due to be shed from the tips of the villi.

Lipid emulsions were prepared with compositions similar to the triacylglycerol-rich plasma lipoproteins, but also incorporating added small amounts of monoacylglycerols. Control emulsions without monoacylglycerol were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. The emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the bloodstream, with the removal rates of triacylglycerols faster than those of cholesteryl esters. Much of the removed cholesteryl ester was found in the liver, but only a small fraction of the triacylglycerol, consistent with hepatic uptake of the triacylglycerol-depleted remnants of the injected emulsion. Emulsions incorporating added monooleoylglycerol or stearic acid were metabolized similarly. Added 1- or 2-monostearoylglycerol had no effect on triacylglycerol removal from plasma, but the removal rate of cholesteryl esters was decreased and less cholesteryl ester was found in the liver. These effects are similar to those recently described when emulsions and chylomicrons contained triacylglycerols with a saturated acyl chain at the glycerol 2-position, suggesting that saturated monoacylglycerol produced by the action of lipoprotein lipase may cause triacylglycerol-depleted remnant particles to remain in the plasma instead of being rapidly taken up by the liver.

The rate of uptake and esterification of fatty acid by everted intestinal sacs is reduced when micellar media of a non-ionic detergent are compared with bile salt micellar media 1 ( Hoffman , Biochim. Biophys. Acta , 196 (1970) 193). In this paper it has been shown that: 1. (1) The reduced rate of uptake correlates with a reduced diffusion coefficient of fatty acid in the non-ionic detergent micellar solution. 2. (2) The sac preparation has a maximal esterifying capacity. 3. (3) This is the same for non-ionic detergent and bile salt micellar media.

1. 1. Individuals of the Australian marsupial species, the possum ( Trichosurus vulpecula ), differ in erythrocyte cation concentration. ATPase activity was measured in the fragmented membranes of erythrocytes in which potassium predominates (HK cells) and in which sodium predominates (LK cells). A comparison was made with erythrocyte membrane ATPase activity in the marsupial quokka ( Setonyx brachyurus ) in which sodium and potassium are present in approximately equal concentration. 2. 2. All erythrocyte preparations showed basal ATPase activity. Significant (Na + -K + )-activated ATPase was present only in HK possum and quokka cells, absolute values and values relative to basal ATPase being higher in HK possum than in quokka cells. 3. 3. These results suggest that the low erythrocyte potassium concentrations in LK possums may be due to the absence of (Na + -K + )-activated ATPase and pump activity in their cell membranes.

Absorption of two lipids from a mixture was compared in unanaesthetized bile diverted rats. The isotopically labelled lipids, oleic acid and glyceryl-1-monoether, were infused intraduodenally in bile salts either as a micellar solution or in an emulsion with no micellar phase present. The two isotopes were absorbed equally into the thoracic duct lymph from micellar solutions. The absorption of both was depressed in the absence of micelles with the monoether label being more affected than the fatty acid label. This suggested a rate-limiting step in the absorptive pathway prior to the appearance of lipid in the lymph. To further localize this step animals were sacrificed at 4 and 6 h and isotope recovered from intestinal lumen and wall as well as lymph. This showed that there was no accumulation of isotope in the intestinal wall but that there was accumulation in the intestinal lumen, suggesting the rate-limiting step to be in the luminal phase of absorption.